RESUMEN
Toll-like receptor 4 (TLR4) has been identified as a potentially promising therapeutic target in acute pancreatitis (AP). However, the role of intestinal TLR4 in AP and AP-associated gut injury remains unclear. This study aimed to explore the relationship between intestinal TLR4 and gut microbiota during AP. A mouse AP model was establish by intraperitoneal injection of L-arginine. Pancreatic injury and intestinal barrier function were evaluated in wild-type and intestinal epithelial TLR4 knockout (TLR4ΔIEC) mice. Gut microbiota was analyzed by 16S rRNA sequencing. Quadruple antibiotics were applied to induce microbiota-depleted mice. Differentially expressed genes in gut were detected by RNA sequencing. L. reuteri treatment was carried out in vivo and vitro study. Compared with wild-type mice, AP and AP-associated gut injury were exacerbated in TLR4ΔIEC mice in a gut microbiota-dependent manner. The relative abundance of Lactobacillus and number of Paneth cells remarkably decreased in TLR4ΔIEC mice. The KEGG pathway analysis derived from RNA sequencing suggested that genes affected by intestinal TLR4 deletion were related to the activation of nod-like receptor pathway. Furthermore, L. reuteri treatment could significantly improve the pancreatic and intestinal injury in TLR4ΔIEC mice through promoting Paneth cells in a NOD2-dependent manner. Loss of intestinal epithelial TLR4 exacerbated pancreatic and intestinal damage during AP, which might be attributed to the gut microbiota dysbiosis especially the exhausted Lactobacillus. L. reuteri might maintain intestinal homeostasis and alleviate AP via Paneth cells modulation.Abbreviations: AP Acute pancreatitis, TLR4 Toll-like receptor 4, IL-1ß Interleukin-1ß, IL-6 Interleukin-6, TNF-α Tumor necrosis factor-α, SIRS Systematic inflammatory response syndrome, LPS Lipopolysaccharides, SPF Specific pathogen-free, ZO-1 Zonula occludens-1, CON Control, H&E Hematoxylin and eosin, FISH Fluorescence in situ hybridization, DAPI 4',6-diamidino-2-phenylindole, PCoA Principal co-ordinates analysis, SCFA Short chain fatty acid, LEfSe Linear discriminant analysis Effect Size, ANOVA Analysis of variance, F/B Firmicutes/Bacteroidetes, PCA Principal component analysis, NOD2 Nod-like receptor 2, ABX antibiotics, PCNA proliferating cell nuclear antigen.
Asunto(s)
Microbioma Gastrointestinal , Pancreatitis , Receptor Toll-Like 4 , Enfermedad Aguda , Animales , Antibacterianos , Disbiosis , Microbioma Gastrointestinal/fisiología , Lactobacillus/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas NLR , Células de Paneth/metabolismo , ARN Ribosómico 16S , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The mammalian gut is inhabited by a massive and complicated microbial community, in which the host achieves a stable symbiotic environment through the interdependence, coordination, reciprocal constraints and participation in an immune response. The interaction between the host gut and the microbiota is essential for maintaining and achieving the homeostasis of the organism. Consequently, gut homeostasis is pivotal in safeguarding the growth and development and potential productive performance of the host. As metabolites of microorganisms, short chain fatty acids are not only the preferred energy metabolic feedstock for host intestinal epithelial cells, but also exert vital effects on antioxidants and the regulation of intestinal community homeostasis. Herein, we summarize the effects of intestinal microorganisms on the host gut and the mechanisms of action of short chain fatty acids on the four intestinal barriers of the organism, which will shed light on the manipulation of the intestinal community to achieve precise nutrition for specific individuals and provide a novel perspective for the prevention and treatment of diseases.
RESUMEN
BACKGROUND/AIMS: Parathyroid hormone-related protein (PTHrP) is implicated in regulating calcium homeostasis in vertebrates, including sea bream, chick, and mammals. However, the molecular mechanism underlying the function of PTHrP in regulating calcium transport is still not fully investigated. This study aimed to investigate the effect of PTHrP on the calcium uptake and its underlying molecular mechanism in rat enterocytes. METHODS: The rat intestinal epithelial cell line (IEC-6) was used. Calcium uptake was determined by using the fluo-4 acetoxymethyl ester fluorescence method. The expression levels of RNAs and proteins was assessed by RT-PCR and Western-blot, respectively. RESULTS: PTHrP (1-40) induced rapid calcium uptake in enterocytes in a dose-dependent manner. PTHrP (1-40) up-regulated parathyroid hormone 1 receptor (PTHR1) protein but not 1,25D3-MARRS receptor. Pre-treatment of anti- PTHR1 antibody abolished the PTHrP (1-40)-induced calcium uptake. PTHrP (1-40) significantly up-regulated four transcellular calcium transporter proteins, potential vanilloid member 6 (TRPV6), calbindin-D9k (CaBP-D9k), sodium-calcium exchanger 1 (NCX1) and plasma membrane calcium ATPase 1 (PMCA1), in a dose- and time-dependent manner. Pre-treatment with TRPV6 or CaBP-D9k antibodies or knockout of rat TRPV6 or CaBP-D9k markedly inhibited PTHrP (1-40)-induced calcium uptake, whereas inhibition of NCX or PMCA1 by antibodies or inhibitors had no effect on PTHrP(1-40)-induced calcium uptake. Furthermore, PTHrP (1-40) treatment up-regulated protein levels of protein kinase C (PKC α/ß) and protein kinase A (PKA). Pretreatment of PKC α/ß inhibitor (but not PKA inhibitor) inhibited PTHrP (1-40)-induced calcium uptake. CONCLUSION: PTHrP (1-40) stimulates calcium uptake via PTHR1 receptor and PKC α/ß signaling pathway in rat enterocytes, and calcium transporters TRPV6 and CaBP-D9k are necessary for this stimulatory effect.
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Calcio/metabolismo , Enterocitos/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/metabolismo , Proteína Quinasa C beta/metabolismo , Proteína Quinasa C-alfa/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Transducción de Señal , Animales , Línea Celular , RatasRESUMEN
BACKGROUND: Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop a vaccine in order to interrupt transmission from animals to humans. A 31 kDa protein from T. spiralis (Ts31) is an antigen targeted by protective antibodies, and Ts31 contains a domain of trypsin-like serine protease that might have the function of serine protease. The purpose of this study was to investigate the molecular characteristics of Ts31 and its induced immune protection. METHODS: Expression and localization of Ts31 in various T. spiralis phases were investigated using qPCR and immunofluorescent test (IFT). The specific binding between Ts31 and intestinal epithelium cells (IECs) was analyzed by Far-Western blotting, ELISA and IFT, and the cellular localization of binding sites was examined on confocal microscopy. The mice were subcutaneously vaccinated with recombinant Ts31 protein (rTs31), serum specific IgG was determined by ELISA, and immune protection induced by immunization with rTs31 was evaluated. Inhibition of anti-rTs31 IgG on IL1 invasion of IECs and ADCC-mediated killing of newborn larvae (NBL) was also determined. RESULTS: Ts31 was expressed at different life-cycle stages and located principally at the stichosome and cuticle of this parasite. rTs31 was capable to specially bond to IECs, and binding site was located in the cytoplasm of IECs. Immunization of mice with rTs31 elicited a significant humoral response and protection, as demonstrated by a 56.93% reduction of adult worms at 6 days post-infection (dpi) and a 53.50% reduction of muscle larvae at 42 dpi after larval challenge. Anti-rTs31 antibodies impeded T. spiralis penetration of enterocytes in a dose-dependent pattern, and participated in the destruction of NBL by an ADCC-mediated manner. CONCLUSIONS: Ts31 facilitated the T. spiralis penetration of intestinal epithelium, which could make it a vaccine candidate target molecule against Trichinella infection.
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Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Trichinella spiralis/inmunología , Triquinelosis/inmunología , Vacunas/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos Helmínticos/genética , Antígenos Helmínticos/metabolismo , Células Cultivadas , Femenino , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Inmunización , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitología , Larva/inmunología , Larva/fisiología , Ratones , Ratones Endogámicos BALB C , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Transcripción Genética , Triquinelosis/metabolismo , Triquinelosis/prevención & control , Vacunas/genética , Vacunas/metabolismoRESUMEN
Overexpression of 5-hydroxytryptamine (5-HT) in human cancer contributes to tumor metastasis, but the role of 5-HT receptor family in cancer has not been thoroughly explored. Here, we report overexpression of 5-HT(1D) receptor (5-HT(1D)R) was associated with Wnt signaling pathway and advanced tumor stage. The underlying mechanism of 5-HT(1D)R-promoted tumor invasion was through its activation on the Axin1/ß-catenin/MMP-7 pathway. In an orthotopic colorectal cancer mouse model, we demonstrated that a 5-HT(1D)R antagonist (GR127935) effectively inhibited tumor metastasis through targeting Axin1. Furthermore, in intestinal epithelium cells, we observed that 5-HT(1D)R played an important role in cell invasion via Axin1/ß-catenin/MMP-7 pathway. Together, our findings reveal an essential role of the physiologic level of 5-HT(1D)R in pulmonary metastasis of colorectal cancer.